Method of synthesizing near IR, closed chain sulfo-cyanine dyes

ABSTRACT

Compositions and methods of synthesizing near IR, closed chain, sulfo-cyanine dyes are provided.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a Divisional of U.S. patent application Ser.No. 15/204,441 filed Jul. 7, 2016 (Allowed), the full disclosure ofwhich is incorporated herein by reference in its entirety for allpurposes.

BACKGROUND OF THE INVENTION

Minimally invasive medical techniques are intended to reduce the amountof tissue that is damaged during diagnostic or surgical procedures,thereby reducing patient recovery time, discomfort, and deleterious sideeffects. While millions of “open” or traditional surgeries are performedeach year in the United States; many of these surgeries can potentiallybe performed in a minimally invasive manner. One effect of minimallyinvasive surgery, for example, is reduced post-operative recovery timeand related hospital stay. Because the average hospital stay for astandard surgery is typically significantly longer than the average stayfor an analogous minimally invasive surgery, increased use of minimallyinvasive techniques could save millions of dollars in hospital costseach year. While many of the surgeries performed in the United Statescould potentially be performed in a minimally invasive manner, only aportion currently employ these techniques due to instrument limitations,method limitations, and the additional surgical training involved inmastering the techniques.

Minimally invasive tele-surgical systems are being developed to increasea surgeon's dexterity as well as to allow a surgeon to operate on apatient from a remote location. Telesurgery is a general term forsurgical systems where the surgeon uses some form of remote control,e.g., a servomechanism, or the like, to manipulate surgical instrumentmovements rather than directly holding and moving the instruments byhand. In such a telesurgery system, the surgeon is provided with animage of the surgical site at the remote location. While viewing thesurgical site on a suitable viewer or display, the surgeon performs thesurgical procedures on the patient by manipulating master control inputdevices, which in turn control the motion of instruments. These inputdevices can move the working ends of the surgical instruments withsufficient dexterity to perform intricate surgical tasks.

Minimally invasive medical techniques, including tele-surgical systemscan be further aided by improving visualization of the tissue where theprocedure is to be carried out. One way to improve visualization oftissue is through the use of dyes capable of targeted visualization oftissue. Thus, there is a need for novel methods of preparing dyescapable of targeted visualization of tissue such as near IR, closedchain, sulfo-cyanine dyes. Surprisingly, the present invention meetsthese and other needs.

BRIEF SUMMARY OF THE INVENTION

The present invention generally provides novel, compositions and methodsof preparing near IR, closed chain, sulfo-cyanine dyes.

In a first embodiment, the present invention provides a method ofpreparing a compound of Formula V:

-   -   or salts thereof,        the method comprising:

forming a reaction mixture comprising a base, a solvent, a compound ofFormula I:

and

-   -   a compound of Formula II:

under conditions suitable to prepare the compound of Formula V having apurity of at least 75%, wherein R¹ is C₁₋₆ alkylene-SO₃H; R² and R³ areeach independently selected from the group consisting of H, C₁₋₆ alkyl,C₁₋₆ alkylene-SO₃H and C₁₋₆ alkylene-COOH; each R⁴ is independentlyselected from the group consisting of H, C₁₋₆ alkyl, —SO₃H, and C₁₋₆alkylene-SO₃H; and subscript n is an integer from 0 to 4.

In a second embodiment, the present invention provides a method ofpreparing a compound of Formula VI:

or salts thereof,the method comprising: forming a reaction mixture comprising a base, asolvent, a compound of Formula V:

anda compound of Formula III:

under an inert atmosphere and without exposure to visible light, toprepare the compound of Formula VI, wherein R¹ and R^(1a) are eachindependently C₁₋₆ alkylene-SO₃H; R², R^(2a), and R³ are eachindependently selected from the group consisting of H, C₁₋₆ alkyl, andC₁₋₆ alkylene-SO₃H; R^(3a) is C₁₋₆ alkylene-COOH; each R⁴ and R^(4a) isindependently selected from the group consisting of H, C₁₋₆ alkyl,—SO₃H, and C₁₋₆ alkylene-SO₃H; and each subscript n is an integer from 0to 4.

In a third embodiment, the present invention provides a compound havingthe formula compound of Formula VI:

wherein R¹ and R^(1a) are each independently C₁₋₆ alkylene-SO₃H; R²,R^(2a), and R³ are each C₁₋₆ alkyl; R^(3a) is C₁₋₆ alkylene-COOH; eachR⁴ and R^(4a) is independently selected from the group consisting of Hand C₁₋₆ alkyl; and each subscript n is an integer from 0 to 4, or saltsthereof.

In a forth embodiment, the present invention provides a method ofpreparing a compound of Formula VII:

or salts thereof, the method comprising: forming a reaction mixturecomprising a base, a solvent, a compound of Formula VI:

anda compound having the structure:

under an inert atmosphere and without exposure to visible light, toprepare the compound of Formula VII, wherein R¹ and R^(1a) are eachindependently C₁₋₆ alkylene-SO₃H; R², R^(2a), and R³ are eachindependently selected from the group consisting of H, C₁₋₆ alkyl, andC₁₋₆ alkylene-SO₃H; R^(3a) is C₁₋₆ alkylene-COOH; each R⁴ and R^(4a) isindependently selected from the group consisting of H, C₁₋₆ alkyl,—SO₃H, and C₁₋₆ alkylene-SO₃H; R⁵ is SO₃H; and each subscript n is aninteger from 0 to 4.

In a fifth embodiment, the present invention provides a compound ofFormula VII:

wherein R¹ and R^(1a) are each independently C₁₋₆ alkylene-SO₃H; R²,R^(2a), and R³ are each independently selected from the group consistingof H, C₁₋₆ alkyl, and C₁₋₆ alkylene-SO₃H; R^(3a) is C₁₋₆ alkylene-COOH;each R⁴ and R^(4a) is independently selected from the group consistingof H, C₁₋₆ alkyl, —SO₃H, and C₁₋₆ alkylene-SO₃H; R⁵ is SO₃H; and eachsubscript n is an integer from 0 to 4, or salts thereof.

In a seventh embodiment, the present invention provides a method ofpreparing a compound of Formula VIII:

the method comprising: forming a reaction mixture comprising a solvent,sodium acetate and a compound of Formula VII having the structure:

under conditions suitable to form the compound of Formula VIIIsubstantially free of the compound having the structure:

In an eighth embodiment, the present invention provides a compoundhaving the structure:

substantially free of the compound having the structure:

In a ninth embodiment, the present invention provides a method ofpreparing a compound of Formula VIII having the structure:

the method comprising:forming a first reaction mixture comprising triethylamine, methanol, acompound of Formula I having the structure:

anda compound of Formula II having the structure:

under conditions sufficient to prepare a compound of Formula V havingthe structure:

or salts thereof,the compound of Formula V having a purity of at least 95% andsubstantially free of a compound of Formula IV having the structure:

forming a second reaction mixture comprising triethylamine, acetic acid,acetonitrile, the compound of Formula V, and a compound of Formula IIIhaving the structure:

under an Argon atmosphere and without exposure to visible light, toprepare the compound of Formula VI having the structure:

or salts thereof;forming a third reaction mixture comprising sodium carbonate,dimethylformamide, the compound of Formula VI, and the compound havingthe structure:

under an inert atmosphere and without exposure to visible light, toprepare the compound of Formula VII having the structure:

andforming a fourth reaction mixture comprising sodium acetate, thecompound of Formula VII, and water, under conditions suitable to formthe compound of Formula VIII substantially free of the compound havingthe structure:

DETAILED DESCRIPTION OF THE INVENTION I. General

The present invention provides novel compositions and methods forpreparing near IR, closed chain, sulfo-cyanine dyes for use invisualizing tissue under illumination with near-infrared radiation.Methods include mono-additions, which allow controlled step-wisecondensations to afford the dyes of the present invention.

II. Definitions

“Forming a reaction mixture” refers to the instance of mixing andreacting two or more substances together causing at least one reactionresulting in a chemical transformation or change.

“Base” refers to a substance that can accept protons or any chemicalcompound that yields hydroxide ions in solution. It is also commonlyreferred to as any substance that can react with an acid to decrease orneutralize its acidic properties, react with acids to form salts, andpromote certain chemical reactions. Examples of bases includenon-nucleophilic bases, amine bases, carbonates, halides, phosphates,hydroxides, disilylamides, and hydrides. The base chosen for aparticular conversion depends on the nature of the starting materials,the solvent or solvents in which the reaction is conducted, and thetemperature at which the reaction is conducted.

“Non-nucleophilic base” refers to a chemical compound that functions asa base with no nucleophilicity. Preferably, the non-nucleophilic basedoes not react with the other compounds and reagents. A variety ofnon-nucleophilic bases are known to those of skill in the art. See,e.g., Richard C. Larock, in “Comprehensive Organic Transformation”,2^(nd) edition, 1999. In some embodiments, the non-nucleophilic base isa tertiary amine. In one example, the tertiary amine is an aliphaticamine. In some embodiments, the tertiary amine is an aromatic amine. Insome embodiments, the tertiary amine is a trialkylamine such astriethylamine or diisopropylethylamine.

“Amine bases” refers to primary, secondary, or tertiary amines,compounds of the formula R′R″R′″N where R′, R″, and R′″ can be hydrogenor organic substituents. Alkylamines where one or more of thesubstituents is an aliphatic group can be used. Examples includeoctylamine, dipentylamine, triethylamine, diisopropylamine, anddiisopropylethylamine, di-isopropyl ethyl amine, trimethylamine,quinuclidine, and tributylamine. Cycloalkylamines where one or more ofthe organic substituents is an alicyclic group such as cyclopropyl,cyclopentyl, or cyclooctyl. Monoaryl amines wherein the nitrogen isdirectly attached to an aromatic ring structure, which can have organicsubstituents, can also be used. Examples include N,N-methylphenylamine,aniline, and 4-methylaniline. Heterocyclic and substituted heterocyclicamines in which the amine nitrogen is incorporated into a ring structuresuch as in pyridine, pyrrolidine, and piperdine can also be used. Otherexamples of amines include imidazole, pyridazine, pyrimidine, andpyrazine and bicyclic amines such as 1,5-Diazabicyclo[4.3.0]non-5-ene(DBN) and 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU).

Other bases useful in the present invention include carbonate salts suchas potassium carbonate, potassium bicarbonate, sodium acetate, sodiumcarbonate, sodium bicarbonate, and cesium carbonate; halides includingcesium fluoride; phosphates such as potassium phosphate, potassiumdihydrogen phosphate, and potassium hydrogen phosphate; hydroxides suchas lithium hydroxide, sodium hydroxide, and potassium hydroxide;disilylamides such as lithium hexamethyldisilazide, potassiumhexamethyldisilazide, and sodium hexamethyldisilazide; and hydrides suchas lithium hydride, sodium hydride, and potassium hydride.

“Solvent” refers to polar, aprotic, protic, and non-polar solvents.Examples of solvents include compounds such as hexanes, benzene,toluene, diethyl ether, chloroform, methylene chloride, ethyl acetate,1,4-dioxane, water, tetrahydrofuran (THF), acetone, acetonitrile, DMF,DMSO, acetic acid, n-butanol, isopropanol, n-propanol, ethanol,methanol, formic acid, carbon tetrachloride, benzenethiol,chlorobenzene, cyclohexanethiol, 1-diethylaminoethanol, ethylenedichloride, ethylene glycol, xylene, 1,1,2,2-tetrachloroethane, phenol,acetic acid, 1-butanol, 2-butanol, 2-butanone, diglyme, dimethylether,dioxane, petroleum ether, N-methyl-2-pyrrolidinone (NMP), heptane,glycerin, HMPA (Hexamethylphosphorus triamide), MTBE (methyl t-butylether), nitromethane, pyrideine, 1-propanol, 2-propanol, andtriethylamine.

“Alkyl” refers to a straight or branched, saturated, aliphatic radicalhaving the number of carbon atoms indicated. For example, C₁-C₆ alkylincludes, but is not limited to, methyl, ethyl, propyl, isopropyl,butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, etc.Other alkyl groups include, but are not limited to heptyl, octyl, nonyl,decyl, etc. Alkyl can include any number of carbons, such as 1-2, 1-3,1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 3-4, 3-5, 3-6,4-5, 4-6 and 5-6. The alkyl group is typically monovalent, but can bedivalent, such as when the alkyl group links two moieties together.

“Alkylene” refers to an alkyl group, as defined above, linking at leasttwo other groups, i.e., a divalent hydrocarbon radical. The two moietieslinked to the alkylene can be linked to the same atom or different atomsof the alkylene. For instance, a straight chain alkylene can be thebivalent radical of —(CH₂)_(n), where n is 1, 2, 3, 4, 5 or 6. Alkylenegroups include, but are not limited to, methylene, ethylene, propylene,isopropylene, butylene, isobutylene, sec-butylene, pentylene andhexylene. Additionally, alkenylene, alkynylene and cycloalkylene aredivalent radicals of alkenyl, alkynyl and cycloalkyl.

“Salts” refers to acid or base salts of the compounds used in themethods of the present invention. Illustrative examples ofpharmaceutically acceptable salts are mineral acid (hydrochloric acid,hydrobromic acid, phosphoric acid, and the like) salts, organic acid(acetic acid, propionic acid, glutamic acid, citric acid and the like)salts, quaternary ammonium (methyl iodide, ethyl iodide, and the like)salts. It is understood that the pharmaceutically acceptable salts arenon-toxic. Additional information on suitable pharmaceuticallyacceptable salts can be found in Remington's Pharmaceutical Sciences,17th ed., Mack Publishing Company, Easton, Pa., 1985, which isincorporated herein by reference.

“Precipitated” and “precipitation” refers to the creation of a solidfrom a solution. When the reaction occurs in a liquid solution, thesolid formed is called the ‘precipitate’.

“Inert atmosphere” refers to an atmosphere either containing less than5,000, in some embodiments, no more than 4,000, or, in yet otherembodiments, 400 to 4,000 parts per million of oxygen. In someembodiments, the inert atmosphere contains no more than 1,000, such as300 to 1,000, parts per million of oxygen. Gases such as nitrogen,argon, carbon dioxide, noble gases, or mixtures thereof are often themajor components of the inert atmosphere, although other non-reactivegases may be used. In certain embodiments, argon is employed for thispurpose.

“Without exposure to visible light” refers to the absence of lighthaving wavelength(s) between about 400 nm and about 750 nm.

“Substantially free” refers to preferred negative limitations of thecompositions of the present invention, and are directed to the amount orconcentration of undesired compounds. Generally, the compositionspreferably contain less than 5%, preferably less than 2%, morepreferably less than 1%, even more preferably less than 0.5%, mostpreferably zero percent of such undesired compounds by weight of thecomposition.

III. Methods for Preparing Formula V

As discussed above, there is a need for novel compositions and methodsof preparing dyes capable of targeted visualization of tissue such asnear IR, closed chain, sulfo-cyanine dyes. The present inventiongenerally provides novel compositions and methods of preparing near IR,closed chain, sulfo-cyanine dyes. As demonstrated below, themono-addition of compounds of Formula I to compounds of Formula II is animportant reaction that allows for a stepwise controlled condensation.

In some embodiments, the present invention provides a method ofpreparing a compound of Formula V:

or salts thereof,the method comprising:

forming a reaction mixture comprising a base, a solvent, a compound ofFormula I:

and

-   -   a compound of Formula II:

under conditions suitable to prepare the compound of Formula V having apurity of at least 75%, wherein R¹ is C₁₋₆ alkylene-SO₃H; R² and R³ areeach independently selected from the group consisting of H, C₁₋₆ alkyl,C₁₋₆ alkylene-SO₃H and C₁₋₆ alkylene-COOH; each R⁴ is independentlyselected from the group consisting of H, C₁₋₆ alkyl, —SO₃H, and C₁₋₆alkylene-SO₃H; and subscript n is an integer from 0 to 4.

Bases useful in the present invention include non-nucleophilic bases,amine bases, carbonates, halides, phosphates, hydroxides, disilylamides,and hydrides. The base chosen for a particular conversion depends on thenature of the starting materials, the solvent or solvents in which thereaction is conducted, and the temperature at which the reaction isconducted.

A variety of non-nucleophilic bases are useful in the present inventionand known to those of skill in the art. See, e.g., Richard C. Larock, in“Comprehensive Organic Transformation,” 2nd edition, 1999. In someembodiments, the non-nucleophilic base is a tertiary amine. In someembodiments, the tertiary amine is an aliphatic amine. In someembodiments, the tertiary amine is an aromatic amine. In someembodiments, the tertiary amine is a trialkylamine such as triethylamineor diisopropylethylamine.

Amine bases useful in the present invention include primary, secondary,or tertiary amines, compounds of the formula R′R″R′″N where R′, R″, andR′″ can be hydrogen or organic sub stituents. Alkylamines where one ormore of the substituents is an aliphatic group can be used. Examplesinclude octylamine, dipentylamine, triethylamine, diisopropylamine, anddiisopropylethylamine, di-isopropyl ethyl amine, trimethylamine,quinuclidine, and tributylamine. Cycloalkylamines where one or more ofthe organic substituents is an alicyclic group such as cyclopropyl,cyclopentyl, or cyclooctyl. Monoaryl amines wherein the nitrogen isdirectly attached to an aromatic ring structure, which can have organicsubstituents, can also be used. Examples include N,N-methylphenylamine,aniline, and 4-methylaniline. Heterocyclic and substituted heterocyclicamines in which the amine nitrogen is incorporated into a ring structuresuch as in pyridine, pyrrolidine, and piperdine can also be used. Otherexamples of amines include imidazole, pyridazine, pyrimidine, andpyrazine and bicyclic amines such as 1,5-Diazabicyclo[4.3.0]non-5-ene(DBN) and 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU).

In some embodiments, the base is a non-nucleophilic base. In someembodiments, the non-nucleophilic base is selected from the groupconsisting of triethylamine, di-isopropyl ethyl amine, quinuclidine,pyridine, sodium acetate, sodium carbonate, potassium carbonate andcesium carbonate. In some embodiments, the base is trimethylamine.

Solvents useful in the present invention include polar, aprotic, protic,and non-polar solvents. Examples of solvents useful in the presentinvention include hexanes, benzene, toluene, diethyl ether, chloroform,methylene chloride, ethyl acetate, 1,4-dioxane, water, tetrahydrofuran(THF), acetone, acetonitrile, DMF, DMSO, acetic acid, n-butanol,isopropanol, n-propanol, ethanol, methanol, formic acid, carbontetrachloride, benzenethiol, chlorobenzene, cyclohexanethiol,1-diethylaminoethanol, ethylene dichloride, ethylene glycol, xylene1,1,2,2-tetrachloroethane, phenol, acetic acid, 1-butanol, 2-butanol,2-butaone, diglyme, dimethylether, dioxane, petroleum ether, (NMP)N-methyl-2-pyrrolidinone, heptane, glycerin, HMPA (Hexamethylphosphorustriamide), MTBE (methyl t-butyl ether), nitromethane, pyrideine,1-propanol, 2-propanol, and triethylamine.

In some embodiments, the solvent is selected from the group consistingof methanol, ethanol, isopropanol, diethyl ether, acetonitrile, andtetrahydrofuran. In some embodiments, the solvent is methanol.

In some embodiments, the compounds of Formula V are prepared at a purityof at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%. In someembodiments, the compound of Formula V is prepared with a purity of atleast 90%. In some embodiments, the compound of Formula V is preparedwith a purity of at least 95%. In some embodiments, the compound ofFormula V is prepared with a purity of at least 98%. In someembodiments, the compound of Formula V is prepared with a purity of atleast 98.8%.

In some embodiments, mono-additions allow for controlled step-wisecondensations. In some embodiments, the compound of Formula V isprepared with less than 25% of a compound of Formula IV being present:

In some embodiments, the compound of Formula V is prepared substantiallyfree of the compound of Formula IV.

In some embodiments, the compound of Formula V is prepared in an amountof at least about 10 g. In some embodiments, the compound of Formula Vis prepared in an amount of at least about 40 g. In some embodiments,the compound of Formula V is prepared in an amount of at least about 70g.

In some embodiments, the method comprises: forming the reaction mixturecomprising triethylamine and/or methanol, the compound of Formula Ihaving the structure:

andthe compound of Formula II having the structure:

under conditions sufficient to prepare the compound of Formula V havingthe structure:

having a purity of at least 95%, substantially free of the compound ofFormula IV having the structure:

or salts thereof.

The method of preparing the compound of Formula V occurs under suitablereaction conditions. Suitable reaction conditions include all reactionconditions suitable for preparing compounds of Formula V. In someembodiments, reaction conditions include reagents, temperature,pressure, and time. One of skill in the art will appreciate that changesand modifications to the reaction conditions may be practiced within thescope of the appended claims.

The reaction mixtures of the method for preparation of Formula V can beat any suitable temperature. For example, the temperature of thereaction mixture can be of from about 0° C. to about 200° C., such as atabout 20, 25, 30, 35, 40, 45, 50, 55, 60, 62, 64, 65, 66, 70, 75 orabout 80° C. In some embodiments, the temperature of the reactionmixture can be from about 25° C. to about 75° C., or of from about 40°C. to about 70° C., or of from about 60° C. to about 70° C. In someembodiments, the temperature of the reaction mixture can be about 64° C.

The reaction mixtures for preparation of Formula V of the method can beat any suitable pressure. For example, the reaction mixture can be atatmospheric pressure or above atmospheric pressure. Pressures greaterthan atmospheric pressure can be achieved by using a pressure vessel andpressurizing with a suitable gas, or using a closed vessel that is thenheated. The reaction mixtures can be also be exposed to any suitableenvironment, such as atmospheric gases, or inert gases such as nitrogenor argon. In some embodiments, the inert gas exposed to the reactionmixture is argon.

The reaction mixtures of the method for preparation of Formula V canalso be agitated by any suitable means. For example, the reactionmixtures can be stirred, shaken, vortexed, or others.

Each reaction mixture of the method for preparation of Formula V can bemixed for any suitable period of time from minutes to hours. Forexample, the reaction mixture can be mixed for about 5 minutes, or 10,15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 minutes, or forabout 3, 4, 6, 12, 16, 24, 36 or 48 hours.

IV. Methods for Preparing Formula VI

The present invention also provides the stepwise addition of compoundsof Formula V to compounds of Formula III. Because compounds of Formula Iand Formula III are added to Formula I compounds in different steps,Formula I and Formula III can vary significantly, which allows for awide variety of compounds of Formula VI.

In some embodiments, the present invention provides a method ofpreparing a compound of Formula VI:

or salts thereof,the method comprising: forming a reaction mixture comprising a base, asolvent, a compound of Formula V:

anda compound of Formula III:

under an inert atmosphere and without exposure to visible light, toprepare the compound of Formula VI, wherein R¹ and R^(1a) are eachindependently C₁₋₆ alkylene-SO₃H; R², R^(2a), and R³ are eachindependently selected from the group consisting of H, C₁₋₆ alkyl, andC₁₋₆ alkylene-SO₃H; R^(3a) is C₁₋₆ alkylene-COOH; each R⁴ and R^(4a) isindependently selected from the group consisting of H, C₁₋₆ alkyl,—SO₃H, and C₁₋₆ alkylene-SO₃H; and each subscript n is an integer from 0to 4.

Bases useful in the present invention include non-nucleophilic bases,amine bases, carbonates, halides, phosphates, hydroxides, disilylamides,and hydrides. The base chosen for a particular conversion depends on thenature of the starting materials, the solvent or solvents in which thereaction is conducted, and the temperature at which the reaction isconducted.

A variety of non-nucleophilic bases are useful in the present inventionand known to those of skill in the art. See, e.g., Richard C. Larock, in“Comprehensive Organic Transformation,” 2nd edition, 1999. In someembodiments, the non-nucleophilic base is a tertiary amine. In someembodiments, the tertiary amine is an aliphatic amine. In someembodiments, the tertiary amine is an aromatic amine. In someembodiments, the tertiary amine is a trialkylamine such as triethylamineor diisopropylethylamine.

Amine bases useful in the present invention include primary, secondary,or tertiary amines, compounds of the formula R′R″R′″N where R′, R″, andR′″ can be hydrogen or organic sub stituents. Alkylamines where one ormore of the substituents is an aliphatic group can be used. Examplesinclude octylamine, dipentylamine, triethylamine, diisopropylamine, anddiisopropylethylamine, di-isopropyl ethyl amine, trimethylamine,quinuclidine, and tributylamine. Cycloalkylamines where one or more ofthe organic substituents is an alicyclic group such as cyclopropyl,cyclopentyl, or cyclooctyl. Monoaryl amines wherein the nitrogen isdirectly attached to an aromatic ring structure, which can have organicsubstituents, can also be used. Examples include N,N-methylphenylamine,aniline, and 4-methylaniline. Heterocyclic and substituted heterocyclicamines in which the amine nitrogen is incorporated into a ring structuresuch as in pyridine, pyrrolidine, and piperdine can also be used. Otherexamples of amines include imidazole, pyridazine, pyrimidine, andpyrazine and bicyclic amines such as 1,5-Diazabicyclo[4.3.0]non-5-ene(DBN) and 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU).

In some embodiments, the base is a non-nucleophilic base. In someembodiments, the non-nucleophilic base is selected from the groupconsisting triethylamine, di-isopropyl ethyl amine, quinuclidine,pyridine, sodium acetate, sodium carbonate, potassium carbonate andcesium carbonate. In some embodiments, the base is trimethylamine.

Solvents useful in the present invention include polar, aprotic, protic,and non-polar solvents. Examples of solvents useful in the presentinvention include hexanes, benzene, toluene, diethyl ether, chloroform,methylene chloride, ethyl acetate, 1,4-dioxane, water, tetrahydrofuran(THF), acetone, acetonitrile, DMF, DMSO, acetic acid, n-butanol,isopropanol, n-propanol, ethanol, methanol, formic acid, carbontetrachloride, benzenethiol, chlorobenzene, cyclohexanethiol,1-diethylaminoethanol, ethylene dichloride, ethylene glycol, xylene,1,1,2,2-tetrachloroethane, phenol, acetic acid, 1-butanol, 2-butanol,2-butanone, diglyme, dimethylether, dioxane, petroleum ether,N-methyl-2-pyrrolidinone (NMP), heptane, glycerin, HMPA(Hexamethylphosphorus triamide), MTBE (methyl t-butyl ether),nitromethane, pyrideine, 1-propanol, 2-propanol, and triethylamine.

In some embodiments, the solvent is selected from the group consistingof acetic acid, methanol, ethanol, isopropanol, diethyl ether,acetonitrile, and THF. In some embodiments, the solvent comprises aceticacid and acetonitrile. In some embodiments, the solvent is acetonitrile.

Inert gases are gases that do not undergo chemical reactions under a setof given conditions. The noble gases often do not react with manysubstances. Inert gases are used generally to avoid unwanted chemicalreactions degrading a sample. These undesirable chemical reactions areoften oxidation and hydrolysis reactions with the oxygen and moisture inair. In some embodiments, the inert atmosphere is Argon.

In some embodiments, R¹ and R^(1a) are each independently C₁₋₆alkylene-SO₃H; R², R^(2a), and R³ are each C₁₋₆ alkyl; R^(3a) is C₁₋₆alkylene-COOH; and each subscript n is 0.

In some embodiments, R¹ and R^(1a) are each C₃ alkylene-SO₃H; R²,R^(2a), and R³ are each C₁₋₂ alkyl; R^(1a) is C₃ alkylene-COOH; and eachsubscript n is 0.

In some embodiments, the method comprises: forming the reaction mixturecomprising triethylamine, acetic acid, acetonitrile, the compound ofFormula V having the structure:

andthe compound of Formula III having the structure:

under an Argon atmosphere and without exposure to visible light, toprepare the compound of Formula VI having the structure:

or salts thereof.

In some embodiments, the compound of Formula V is prepared by the methoddescribed above.

In some embodiments, the present invention provides a compound havingthe formula compound of Formula VI:

wherein R¹ and R^(1a) are each independently C₁₋₆ alkylene-SO₃H; R²,R^(2a), and R³ are each C₁₋₆ alkyl; R^(3a) is C₁₋₆ alkylene-COOH; eachR⁴ and R^(4a) is independently selected from the group consisting of Hand C₁₋₆ alkyl; and each subscript n is an integer from 0 to 4, or saltsthereof.

In some embodiments, the compound of Formula VI has the structure:

or salts thereof.

The method of preparing the compound of Formula VI occurs under suitablereaction conditions. Suitable reaction conditions include all reactionconditions suitable for preparing compounds of Formula VI. In someembodiments, reaction conditions include reagents, temperature,pressure, and time. One of skill in the art will appreciate that changesand modifications to the reaction conditions may be practiced within thescope of the appended claims.

The reaction mixtures of the method for preparation of Formula VI can beat any suitable temperature. For example, the temperature of thereaction mixture can be of from about 0° C. to about 200° C., such as atabout 20, 25, 30, 35, 40, 42, 45, 50, 52, 55, 60, 62, 64, 65, 66, 70, 75or about 80° C. In some embodiments, the temperature of the reactionmixture can be from about 25° C. to about 75° C., or of from about 30°C. to about 60° C., or of from about 40° C. to about 55° C. In someembodiments, the temperature of the reaction mixture can be about 42° C.

The reaction mixtures for preparation of Formula VI of the method can beat any suitable pressure. For example, the reaction mixture can be atatmospheric pressure or above atmospheric pressure. Pressures greaterthan atmospheric pressure can be achieved by using a pressure vessel andpressurizing with a suitable gas, or using a closed vessel that is thenheated. The reaction mixtures can be also be exposed to any suitableenvironment, such as atmospheric gases, or inert gases such as nitrogenor argon. In some embodiments, the inert gas exposed to the reactionmixture is argon.

The reaction mixtures of the method for preparation of Formula VI canalso be agitated by any suitable means. For example, the reactionmixtures can be stirred, shaken, vortexed, or others.

Each reaction mixture of the method for preparation of Formula VI can bemixed for any suitable period of time from minutes to hours. Forexample, the reaction mixture can be mixed for about 5 minutes, or 10,15, 20, 30, 45 or 60 minutes, or for about 1, 2, 3, 4, 6, 12, 16, 24, 36or 48 hours.

V. Methods for Preparing Formula VII

The present invention provides novel, compositions and methods ofpreparing near IR, closed chain, sulfo-cyanine dyes. As demonstratedbelow, compounds of Formula VII are synthesized from compounds ofFormula VI.

In some embodiments, the present invention provides a method ofpreparing a compound of Formula VII:

or salts thereof, the method comprising: forming a reaction mixturecomprising a base, a solvent, a compound of Formula VI:

anda compound having the structure:

under an inert atmosphere and without exposure to visible light, toprepare the compound of Formula VII, wherein R¹ and R^(1a) are eachindependently C₁₋₆ alkylene-SO₃H; R², R^(2a), and R³ are eachindependently selected from the group consisting of H, C₁₋₆ alkyl, andC₁₋₆ alkylene-SO₃H; R^(3a) is C₁₋₆ alkylene-COOH; each R⁴ and R^(4a) isindependently selected from the group consisting of H, C₁₋₆ alkyl,—SO₃H, and C₁₋₆ alkylene-SO₃H; R⁵ is SO₃H; and each subscript n is aninteger from 0 to 4. In some embodiments, R¹ and R^(1a) are eachindependently C₁₋₆ alkylene-SO₃H; R², R^(2a), and R³ are each C₁₋₆alkyl; R^(3a) is C₁₋₆ alkylene-COOH; and R⁵ is SO₃H.

Bases useful in the present invention include non-nucleophilic bases,amine bases, carbonates, halides, phosphates, hydroxides, disilylamides,and hydrides. The base chosen for a particular conversion depends on thenature of the starting materials, the solvent or solvents in which thereaction is conducted, and the temperature at which the reaction isconducted.

A variety of non-nucleophilic bases are useful in the present inventionand known to those of skill in the art. See, e.g., Richard C. Larock, in“Comprehensive Organic Transformation,” 2nd edition, 1999. In someembodiments, the non-nucleophilic base is a tertiary amine. In someembodiments, the tertiary amine is an aliphatic amine. In someembodiments, the tertiary amine is an aromatic amine. In someembodiments, the tertiary amine is a trialkylamine such as triethylamineor diisopropylethylamine.

Amine bases useful in the present invention include primary, secondary,or tertiary amines, compounds of the formula R′R″R′″N where R′, R″, andR′″ can be hydrogen or organic sub stituents. Alkylamines where one ormore of the substituents is an aliphatic group can be used. Examplesinclude octylamine, dipentylamine, triethylamine, diisopropylamine, anddiisopropylethylamine, di-isopropyl ethyl amine, trimethylamine,quinuclidine, and tributylamine. Cycloalkylamines where one or more ofthe organic substituents is an alicyclic group such as cyclopropyl,cyclopentyl, or cyclooctyl. Monoaryl amines wherein the nitrogen isdirectly attached to an aromatic ring structure, which can have organicsubstituents, can also be used. Examples include N,N-methylphenylamine,aniline, and 4-methylaniline. Heterocyclic and substituted heterocyclicamines in which the amine nitrogen is incorporated into a ring structuresuch as in pyridine, pyrrolidine, and piperdine can also be used. Otherexamples of amines include imidazole, pyridazine, pyrimidine, andpyrazine and bicyclic amines such as 1,5-Diazabicyclo[4.3.0]non-5-ene(DBN) and 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU).

In some embodiments, the base is a non-nucleophilic base. In someembodiments, the non-nucleophilic base is selected from the groupconsisting triethylamine, di-isopropyl ethyl amine, quinuclidine,pyridine, sodium acetate, sodium carbonate, potassium carbonate andcesium carbonate. In some embodiments the base is sodium carbonate.

Solvents useful in the present invention include polar, aprotic, protic,and non-polar solvents. Examples of solvents useful in the presentinvention include hexanes, benzene, toluene, diethyl ether, chloroform,methylene chloride, ethyl acetate, 1,4-dioxane, water, tetrahydrofuran(THF), acetone, acetonitrile, dimethylformamide (DMF), DMSO, aceticacid, n-butanol, isopropanol, n-propanol, ethanol, methanol, formicacid, carbon tetrachloride, benzenethiol, chlorobenzene,cyclohexanethiol, 1-diethylaminoethanol, ethylene dichloride, ethyleneglycol, xylene 1,1,2,2-tetrachloroethane, phenol, acetic acid,1-butanol, 2-butanol, 2-butaone, diglyme, dimethylether, dioxane,petroleum ether, (NMP) N-methyl-2-pyrrolidinone, heptane, glycerin, HMPA(Hexamethylphosphorus triamide), MTBE (methyl t-butyl ether),nitromethane, pyrideine, 1-propanol, 2-propanol, and triethylamine.

In some embodiments, the solvent is selected from the group consistingof DMF, acetic acid, methanol, ethanol, isopropanol, diethyl ether,acetonitrile, and tetrahydrofuran. In some embodiments, the solventcomprises acetic acid and acetonitrile. In some embodiments, the solventcomprises DMF.

In some embodiments, the compound of Formula VII is prepared in anamount of at least about 1 g. In some embodiments, the compound ofFormula VII is prepared in an amount of at least about 50 g.

In some embodiments, the compound of Formula VII prepared has thestructure:

In some embodiments, the compound of Formula VI is prepared by themethod described above.

In some embodiments, the method comprises: forming the reaction mixturecomprising sodium carbonate, dimethylformamide, the compound of FormulaVI having the structure:

andthe compound having the structure:

under an inert atmosphere and without exposure to visible light, toprepare the compound of Formula VII having the structure:

or salts thereof.

In some embodiments, the present invention provides a compound ofFormula VII:

wherein R¹ and R^(1a) are each independently C₁₋₆ alkylene-SO₃H; R²,R^(2a), and R³ are each independently selected from the group consistingof H, C₁₋₆ alkyl, and C₁₋₆ alkylene-SO₃H; R^(3a) is C₁₋₆ alkylene-COOH;each R⁴ and R^(4a) is independently selected from the group consistingof H, C₁₋₆ alkyl, —SO₃H, and C₁₋₆ alkylene-SO₃H; R⁵ is SO₃H; and eachsubscript n is an integer from 0 to 4, or salts thereof.

In some embodiments, the compound of Formula VII has the structure:

or salts thereof.

In some embodiments, the compound of Formula VII has the structure:

The method of preparing the compound of Formula VII occurs undersuitable reaction conditions. Suitable reaction conditions include allreaction conditions suitable for preparing compounds of Formula VII. Insome embodiments, reaction conditions include reagents, temperature,pressure, and time. One of skill in the art will appreciate that changesand modifications to the reaction conditions may be practiced within thescope of the appended claims.

The reaction mixtures of the method for preparation of Formula VII canbe at any suitable temperature. For example, the temperature of thereaction mixture can be of from about 0° C. to about 200° C., such as atabout 20, 25, 30, 35, 40, 45, 50, 55, 60, 62, 64, 65, 66, 70, 75 orabout 80° C. In some embodiments, the temperature of the reactionmixture can be from about 25° C. to about 75° C., or of from about 40°C. to about 70° C., or of from about 60° C. to about 70° C. In someembodiments, the temperature of the reaction mixture can be about 62° C.

The reaction mixtures for preparation of Formula VII of the method canbe at any suitable pressure. For example, the reaction mixture can be atatmospheric pressure or above atmospheric pressure. Pressures greaterthan atmospheric pressure can be achieved by using a pressure vessel andpressurizing with a suitable gas, or using a closed vessel that is thenheated. The reaction mixtures can be also be exposed to any suitableenvironment, such as atmospheric gases, or inert gases such as nitrogenor argon. In some embodiments, the inert gas exposed to the reactionmixture is argon.

The reaction mixtures of the method for preparation of Formula VII canalso be agitated by any suitable means. For example, the reactionmixtures can be stirred, shaken, vortexed, or others.

Each reaction mixture of the method for preparation of Formula VII canbe mixed for any suitable period of time from minutes to hours. Forexample, the reaction mixture can be mixed for about 5 minutes, or 10,15, 20, 30, 45 or 60 minutes, or for about 1, 2, 3, 4, 6, 12, 16, 24, 36or 48 hours.

VI. Methods for Formula VIII

The present invention provides novel, compositions and methods ofpreparing near IR, closed chain, sulfo-cyanine dyes. As demonstratedbelow, near IR, closed chain, sulfo-cyanine dyes (compounds of FormulaVIII) are synthesized from compounds of Formula VII.

In some embodiments, the present invention provides a method ofpreparing a compound of Formula VIII:

the method comprising: forming a reaction mixture comprising a solvent,sodium acetate and a compound of Formula VII having the structure:

under conditions suitable to form the compound of Formula VIIIsubstantially free of the compound having the structure:

Solvents useful in the present invention include polar, aprotic, protic,and non-polar solvents. Examples of solvents useful in the presentinvention include hexanes, benzene, toluene, diethyl ether, chloroform,methylene chloride, ethyl acetate, 1,4-dioxane, water, tetrahydrofuran(THF), acetone, acetonitrile, DMF, DMSO, acetic acid, n-butanol,isopropanol, n-propanol, ethanol, methanol, formic acid, carbontetrachloride, benzenethiol, chlorobenzene, cyclohexanethiol,1-diethylaminoethanol, ethylene dichloride, ethylene glycol, xylene1,1,2,2-tetrachloroethane, phenol, acetic acid, 1-butanol, 2-butanol,2-butaone, diglyme, dimethylether, dioxane, petroleum ether, (NMP)N-methyl-2-pyrrolidinone, heptane, glycerin, HMPA (Hexamethylphosphorustriamide), MTBE (methyl t-butyl ether), nitromethane, pyrideine,1-propanol, 2-propanol, and triethylamine.

In some embodiments, the solvent is selected from the group consistingof water, methanol, ethanol, isopropanol, diethyl ether,tetrahydrofuran, acetonitrile, acetone, ethyl acetate, n-heptane,hexanes and cyclohexane. In some embodiments, the solvent compriseswater.

In some embodiments, the method further comprises: washing the reactionmixture with acetonitrile to substantially remove the water.

In some embodiments, the method further comprises: forming a solution ofthe compound of Formula VIII and methanol; adding the solution to ethylacetate to precipitate the compound of Formula VIII; washing theprecipitated compound of Formula VIII with n-heptane; and heating theprecipitated compound of Formula VIII at a temperature of from about 50°C. to about 100° C., thereby forming the compound of Formula VIII havingless than 5000 ppm of solvent.

In some embodiments, the compound of Formula VII is prepared by themethod described above.

In some embodiments, the compound of Formula VIII has the structure:

and is substantially free of the compound having the structure:

In some embodiments, near IR, closed chain, sulfo-cyanine dyes(compounds of Formula VIII) are synthesized from compounds of Formula I,II, and III. Methods include the mono-addition of compounds of Formula Ito compounds of Formula II, an important reaction that allows for astepwise controlled condensation to produce compounds of Formula V.Subsequent condensation of compounds of Formula V and III allow for awide variety of compounds of Formula VI. Compounds of Formula VII canthen be synthesized from compounds of Formula VI, which in turn can betreated with sodium acetate to afford compounds of Formula VIII.

In some embodiments, the method of preparing a compound of Formula VIIIhaving the structure:

the method comprising:forming a first reaction mixture comprising triethylamine, methanol, acompound of Formula I having the structure:

anda compound of Formula II having the structure:

under conditions sufficient to prepare a compound of Formula V havingthe structure:

or salts thereof,the compound of Formula V having a purity of at least 95% andsubstantially free of a compound of Formula IV having the structure:

forming a second reaction mixture comprising triethylamine, acetic acid,acetonitrile, the compound of Formula V, and a compound of Formula IIIhaving the structure:

under an Argon atmosphere and without exposure to visible light, toprepare the compound of Formula VI having the structure:

or salts thereof;forming a third reaction mixture comprising sodium carbonate,dimethylformamide, the compound of Formula VI, and the compound havingthe structure:

under an inert atmosphere and without exposure to visible light, toprepare the compound of Formula VII having the structure:

andforming a fourth reaction mixture comprising sodium acetate, thecompound of Formula VII, and water, under conditions suitable to formthe compound of Formula VIII substantially free of the compound havingthe structure:

The method of preparing the compound of Formula VIII occurs undersuitable reaction conditions. Suitable reaction conditions include allreaction conditions suitable for preparing compounds of Formula VIII. Insome embodiments, reaction conditions include reagents, temperature,pressure, and time. One of skill in the art will appreciate that changesand modifications to the reaction conditions may be practiced within thescope of the appended claims.

The reaction mixtures of the method for preparation of Formula VIII canbe at any suitable temperature. For example, the temperature of thereaction mixture can be of from about 0° C. to about 200° C., such as atabout 0, 5, 10, 15, 20, 22, 25, 27, 30, 35, 40, 45 or about 50° C. Insome embodiments, the temperature of the reaction mixture can be fromabout 10° C. to about 40° C., or of from about 15° C. to about 35° C.,or of from about 20° C. to about 30° C. In some embodiments, thetemperature of the reaction mixture can be about 25° C.

The reaction mixtures for preparation of Formula VIII of the method canbe at any suitable pressure. For example, the reaction mixture can be atatmospheric pressure or above atmospheric pressure. Pressures greaterthan atmospheric pressure can be achieved by using a pressure vessel andpressurizing with a suitable gas, or using a closed vessel that is thenheated. The reaction mixtures can be also be exposed to any suitableenvironment, such as atmospheric gases, or inert gases such as nitrogenor argon. In some embodiments, the inert gas exposed to the reactionmixture is argon.

The reaction mixtures of the method for preparation of Formula VIII canalso be agitated by any suitable means. For example, the reactionmixtures can be stirred, shaken, vortexed, or others.

Each reaction mixture of the method for preparation of Formula VIII canbe mixed for any suitable period of time from minutes to hours. Forexample, the reaction mixture can be mixed for about 5 minutes, or 10,15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 minutes, or forabout 3, 4, 6, 12, 16, 24, 36 or 48 hours.

VII. Examples Example 1. Synthesis of Formula V Compound (Stage 5)

Charge Methanol (1250 ml) into a 3 L RB Flask under argon atmosphere.Charge compound a (50 g) into the above RB flask under argon atmosphere.Stir the mass for 10±5 min at 27±3° C. Charge compound b (76.61 g) intothe above reaction mass at 27±3° C. Charge triethylamine (21.57 g) intothe above reaction mass at 27±6° C. Heat the reaction mass to 64±2° C.and maintain for 90±10 min at 64±2° C.

When the reaction is complete, cool the reaction mass to 43±2° C. anddistil off methanol from the reaction mass to 625±10 mm under vacuum.Cool the reaction mass to 12±2° C. and stir for 1 hr±15 min. Filter theproduct and wash the product using ethyl acetate (750 ml). Suck dry andunload the product and weight it. Wet weight: 49.1 g Charge Methanol(300 ml) in the 1 lit RB flask. Charge wet solid into the above RBflask. Heat the mass to 66±3° C. and maintain for 15±5 min. Cool themass to 25±2° C. and stir for 1 hr. Filter the product and wash theproduct using ethyl acetate (500 ml). Suck dry and unload the productand weight it. [Wet weight: 44.0 g]. Charge Methanol (300 ml) in the 1lit RB flask. Charge wet solid into the above RB flask. Heat the mass to66±3° C. and maintain for 15±5 min. Cool the mass to 25±2° C. and stirfor 1 hr. Filter the product and wash the product using ethyl acetate(500 ml). Dry the product under vacuum at 40±2° C. for 6 hr. Weight:42.0 g (Yield: 46.2%). Purity: 98.8%. ¹H NMR (DMSO-d⁶, ppm): δ 1.70 (s,6H), 4.55 (t, 2H), 2.07 (m, 2H), 2.58 (t, 2H), 7.72 (d, 2H), 7.51 (t,1H), 7.15 (t, 1H), 6.80 (d, 1H), 8.45 (d, 1H), 2.68 (t, 2H), 1.85 (m,2H), 2.75 (t, 2H), 8.19 (s, 1H), 10.18 (s, 1H), 7.42 (d, 5H).

Example 2. Synthesis of Formula VI Compound (Stage 6)

Note: Degas the acetonitrile, acetic acid and triethyl amine with argonindividually for 1 hr at 25±2° C. Compounds of these types are reportedto undergo oxidative cleavage with oxygen in presence of light. Sooxygen and light should be excluded during the reaction. Chargeacetonitrile (380 ml) into a 1000 ml RB flask under dark and argonatmosphere. Purge acetonitrile with argon gas and continue the argonpurging throughout the reaction. Charge compound c (38.0 g) into theabove RB flask at 27±3° C. Charge acetic acid (152 ml) into the abovereaction mass at 27±3° C. Heat the reaction mass to 52±3° C. Chargecompound d (28.0 g) into the above reaction mass at 52±3° C. Chargetriethyl amine (38.0 g) into the above reaction mass at 52±3° C. Coolthe reaction mass to 42±3° C. over the period of 40±5 min. Stir thereaction mass for 3 hr at 42±3° C. Charge slowly diisopropyl ether (1900ml) into the above reaction mass at 27±3° C. over the period of 30±10min. Stir the reaction mass for 30±10 min at 27±3° C. Stop the stirringand settle for 30 min. Decant the diisopropyl ether from the mass.Charge methanol (190 ml) into the above mass and stir for 20±5 min at27±3° C. Charge ethyl acetate (11400 ml) into a 20 L RB flask. Chargeslowly the methanol solution of the crude material into above RB flaskover the period of 40±10 min at 27±3° C. Stir the mass for 40±10 min at27±3° C. Filter the mass under nitrogen atmosphere and wash the productwith ethyl acetate (304 ml). Unload the product and dry the productunder vacuum at 40±3° C. for 6 hr. Weight: 65.0 g (Yield: 94.13%).Purity: 80.2%. ¹H NMR (DMSO-d⁶, ppm): δ 1.68 (s, 6H), 1.65 (s, 3H), 2.75(t, 4H), 6.54 (m, 6H), 4.40 (t, 4H), 6.57 (d, 2H), 8.26 (dd, 2H), 2.59(t, 4H), 2.25 (m, 2H), 1.85 (t, 2H), 0.7-1.1 (d, 2H), 7.2-7.6 (m, 8H),1.1-1.2 (t, 9H), 3.0-3.1 (q, 6H).

Example 3. Synthesis of Formula VII Compound (Stage 7)

Charge DMF (1400 ml) into a 3 L RB flask under argon atmosphere. PurgeDMF with argon for 1 hr. Charge 4-hydroxybenzenesulfonic acid hydrate(122.92 g) into the above RB flask under argon atmosphere. Charge sodiumcarbonate (112.14 g) into the above reaction mass at 27±3° C. Stir themass for 15±5 min at 27±3° C. Charge compound f (140 g) into the abovereaction mass at 27±3° C. Heat the reaction mass to 62±2° C. andmaintain for 2 hrs at 62±2° C.

Cool the reaction mass to 40±2° C. Filter the mass through Bucknerfunnel and wash the solid using DMF (280 ml). Charge the filtrate slowlyinto acetone (2520 ml) over a period of 40±5 min. Stir the mass for 40±5min. Filter the product and wash the product using acetone (560 ml).Unload the wet material in double poly bag.

Charge acetone (2100 ml) into a 5 L RB flask. Charge the wet materialinto the above RB flask. Stir the mass for 1 hr±10 min at 27±3° C.Filter the product and wash the product using acetone (280 ml). Dry theproduct under vacuum at 40±2° C. for 6 hr. Crude weight: 180.0 g. Chargemethanol (1800 ml) into a 5 L RB flask. Charge the crude material intothe above RB flask under stirring. Stir the mass for 15±5 min. Chargesilica gel (230-400 mesh) (540 g) into the above RB flask. Stir the massfor 15±5 min. Concentrate the mass under vacuum (250-10 mbar) at 42±3°C. till no more methanol distils off from the mass. Apply high vacuumand remove the final traces of methanol at 42±3° C. Dry the materialunder high vacuum at 42±3° C. for 4 hrs. Weight: 790 g. Charge silicagel (230-400 mesh, 1800 g) into 10 L beaker.

Charge chloroform (5000 ml) into the above beaker and stir for 10 min.Charge the silica gel slurry into the 10 kg column. Note: Allow theslurry to settle for 12 hrs. Charge the crude compound h slurry into theabove column slowly. Column eluted with 16920 ml solvent mixture ofCHCl₃:MeOH:Aq.NH₃ (8.0:1.9:0.1). Note: Collected fractions to be checkedfor presence of product by TLC. Spot the fractions directly in the TLCplate against the crude material and elute with a mixture ofCHCl₃:MeOH:aq. NH3=6.0:3.8:0.2. Non polar impurities were observed inthe fraction. Column eluted with 112500 ml of solvent mixture ofCHCl₃:MeOH:Aq.NH₃ (7.0:2.85:0.15). Note: Non polar impurities wereobserved in the fraction. Column eluted with 46340 ml of solvent mixtureof CHCl₃:MeOH:Aq.NH3 (6.7:3.1:0.2). Note: Non polar impurities wereobserved in the fraction. Column eluted with 11250 ml of solvent mixtureof CHCl₃:MeOH:Aq.NH₃ (6.5:3.3:0.2). Note: Non polar impurities wereobserved in the fraction. Column eluted with 90000 ml of solvent mixtureof CHCl₃:MeOH:Aq.NH₃ (6.3:3.5:0.2). Column eluted with 135000 ml ofsolvent mixture of CHCl₃:MeOH:Aq.NH₃ (6.0:3.8:0.2) and collected asseven fractions. Take 200 ml from each fraction and concentrate eachfraction separately under vacuum at 42±3° C. to the mass volume about 1ml. Submit the sample of each fraction for HPLC analysis. Combine thefraction having HPLC purity greater than 94.0%. Concentrate the masscompletely under high vacuum at 42±3° C. Charge methanol (300 ml) intothe above mass and stir for 10±5 min. Charge ethyl acetate (3000 ml)into a 5 L RB flask. Charge methanol solution into the above RB flaskcontaining ethyl acetate, slowly over a period of 20±5 min. Stir themass for 1 hr±10 min. Filter the product and wash the product usingethyl acetate (200 ml). Dry the product under high vacuum at 50±5° C.for 6 hrs. Weight: 50.34 g (Yield: 33.29%). Purity: 95.54%. ¹H NMR(DMSO-d⁶, ppm): δ 1.26-1.37 (s, 9H), 4.26-4.34 (dd, 4H), 1.86-2.03 (m,8H), 2.74-2.76 (dd, 4H), 1.64-1.71 (t, 2H), 0.45-0.73 (d, 2H), 2.53-2.57(t, 4H), 7.7-7.86 (dd, 2H), 6.36-6.45 (t, 2H), 7.16-7.52 (m, 8H),7.11-7.13 (d, 2H), 7.63-7.66 (d, 2H).

Example 4. Synthesis of Formula VIII Compound (Stage 8)

Charge millipore water (125 ml) into a 1 L RB Flask. Charge compound h(25 g) into the above RB Flask. Stir the mass for 15±5 min at 27±2° C.Charge anhydrous sodium acetate (4.13 g) into the above reaction at27±3° C. in three lots. Stir the reaction mass for 15±5 min at 27±3° C.Charge acetonitrile (750 ml) into the above reaction mass at 25±3° C.over the period of 20±5 min. Stir the reaction mass for 15±5 min. Stopstirring and settle for 15±5 min. Decant the acetonitrile and collectseparately. Charge acetonitrile (125 ml) and stir for 10±5 min. Decantthe acetonitrile and collect separately. Charge acetonitrile (125 ml)and stir for 10±5 min. Decant the acetonitrile and collect separately.Submit the sticky mass for HPLC analysis. Limit: HPLC purity should beNLT 97.5% (area). If IPC does not comply, charge millipore water (125ml) into the above sticky mass and charge acetonitrile (750 ml) into theabove reaction mass at 25±3° C. over the period of 20±5 min. Stir thereaction mass for 15±5 min. Stop stirring and settle for 15±5 min.Decant the acetonitrile and collect separately. Charge acetonitrile (125ml) and stir for 10±5 min. Decant the acetonitrile and collectseparately. Charge acetonitrile (125 ml) and stir for 10±5 min. Decantthe acetonitrile and collect separately. Submit the sticky mass for HPLCanalysis. Repeat these operations until a HPLC purity NLT 97.5% isachieved. If complies, charge methanol (125 ml) into the sticky mass andstir for 10±5 min. Charge ethyl acetate (1250 ml) into a 3 L RB Flask.Charge methanol mass into the above RB flask over a period of 20±5 min.Stir the mass for 2 hr±10 min at 27±3° C. Filter the product and washthe product using n-heptane (2*125 ml) Unload the material and weigh it.Wet weight: 50 g.

Charge n-heptane (250 ml) into the 500 ml RB flask. Charge the wet solidinto the above RB flask. Stir the mass for 1 hr±10 min at 27±3° C.Filter the product and wash the product using n-heptane (2*50 ml). Drythe product under high vacuum at 85±5° C. for 12 hrs. Weight: 17.4 g(Yield 68.9%). Purity: 97.74%. ¹H NMR (CD₃OD, ppm): δ 1.3-1.33 (s, 6H),1.37 (s, 3H), 2.94-2.98 (t, 4H), 2.16-2.26 (m, 4H), 4.3-4.35 (t, 4H),6.33-6.40 (dd, 2H), 7.90-7.99 (dd, 2H), 2.78-2.82 (t, 4H), 2.0-2.08 (m,2H), 1.74-1.84 (m, 1H), 1.74-1.85 (m, 1H), 0.65-1.08 (br, 2H), 1.89-1.94(m, 2H), 7.17-7.89 (m, 12H).

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, one of skill in the art will appreciate that certainchanges and modifications may be practiced within the scope of theappended claims. In addition, each reference provided herein isincorporated by reference in its entirety to the same extent as if eachreference was individually incorporated by reference. Where a conflictexists between the instant application and a reference provided herein,the instant application shall dominate.

What is claimed is:
 1. A method of preparing a compound of Formula VI:

or salts thereof, the method comprising: forming a reaction mixturecomprising a base, a solvent, a compound of Formula V:

and a compound of Formula III:

under an inert atmosphere and without exposure to visible light, toprepare the compound of Formula VI, wherein R¹ and R^(1a) are eachindependently C₁₋₆ alkylene-SO₃H; R², R^(2a), and R³ are eachindependently selected from the group consisting of H, C₁₋₆ alkyl, andC₁₋₆ alkylene-SO₃H; R^(3a) is C₁₋₆ alkylene-COOH; each R⁴ and R^(4a) isindependently selected from the group consisting of H, C₁₋₆ alkyl,—SO₃H, and C₁₋₆ alkylene-SO₃H; and each subscript n is an integer from 0to
 4. 2. The method of claim 1, wherein the base is a non-nucleophilicbase.
 3. The method of claim 2, wherein the non-nucleophilic base isselected from the group consisting triethylamine, di-isopropyl ethylamine, quinuclidine, pyridine, sodium acetate, sodium carbonate,potassium carbonate and cesium carbonate.
 4. The method of claim 1,wherein solvent comprises acetic acid and acetonitrile.
 5. The method ofclaim 1, wherein the inert atmosphere is Argon.
 6. The method of claim1, wherein R¹ and R^(1a) are each independently C₁₋₆ alkylene-SO₃H; R²,R^(2a), and R³ are each C₁₋₆ alkyl; R^(1a) is C₁₋₆ alkylene-COOH; andeach subscript n is
 0. 7. The method of claim 1, comprising: forming thereaction mixture comprising triethylamine, acetic acid, acetonitrile,the compound of Formula V having the structure:

and the compound of Formula III having the structure:

under an Argon atmosphere and without exposure to visible light, toprepare the compound of Formula VI having the structure:

or salts thereof.
 8. The method of claim 1, wherein the compound ofFormula V is prepared by a non-nucleophilic base.
 9. A method ofpreparing a compound of Formula VII:

or salts thereof, the method comprising: forming a reaction mixturecomprising a base, a solvent, a compound of Formula VI:

and a compound having the structure:

under an inert atmosphere and without exposure to visible light, toprepare the compound of Formula VII, wherein R¹ and R^(1a) are eachindependently C₁₋₆ alkylene-SO₃H; R², R^(2a), and R³ are eachindependently selected from the group consisting of H, C₁₋₆ alkyl, andC₁₋₆ alkylene-SO₃H; R^(3a) is C₁₋₆ alkylene-COOH; each R⁴ and R^(4a) isindependently selected from the group consisting of H, C₁₋₆ alkyl,—SO₃H, and C₁₋₆ alkylene-SO₃H; R⁵ is SO₃H; and each subscript n is aninteger from 0 to
 4. 10. The method of claim 9, wherein the base is anon-nucleophilic base.
 11. The method of claim 10, wherein thenon-nucleophilic base is selected from the group consistingtriethylamine, di-isopropyl ethyl amine, quinuclidine, pyridine, sodiumacetate, sodium carbonate, potassium carbonate and cesium carbonate. 12.The method of claim 9, wherein solvent comprises dimethylformamide. 13.The method of claim 9, wherein R¹ and R^(1a) are each independently C₁₋₆alkylene-SO₃H; R², R^(2a), and R³ are each C₁₋₆ alkyl; R^(3a) is C₁₋₆alkylene-COOH; and R⁵ is SO₃H.
 14. The method of claim 9, wherein thecompound of Formula VII is prepared in an amount of at least about 1 g.15. The method of claim 9, comprising: forming the reaction mixturecomprising sodium carbonate, dimethylformamide, the compound of FormulaVI having the structure:

and the compound having the structure:

under an inert atmosphere and without exposure to visible light, toprepare the compound of Formula VII having the structure:

or salts thereof.
 16. The method of claim 9, wherein the compound ofFormula VI

or salts thereof, is prepared by a the method comprising: forming areaction mixture comprising a base, a solvent, a compound of Formula V:

and a compound of Formula III:

under an inert atmosphere and without exposure to visible light, toprepare the compound of Formula VI, wherein R¹ and R^(1a) are eachindependently C₁₋₆ alkylene-SO₃H; R², R^(2a), and R³ are eachindependently selected from the group consisting of H, C₁₋₆ alkyl, andC₁₋₆ alkylene-SO₃H; R^(1a) is C₁₋₆ alkylene-COOH; each R⁴ and R^(4a) isindependently selected from the group consisting of H, C₁₋₆ alkyl,—SO₃H, and C₁₋₆ alkylene-SO₃H; and each subscript n is an integer from 0to
 4. 17. The method of claim 9, wherein the compound of Formula VII hasthe structure:


18. A method of preparing a compound of Formula VIII:

the method comprising: forming a reaction mixture comprising a solvent,sodium acetate and a compound of Formula VII having the structure:

under conditions suitable to form the compound of Formula VIIIsubstantially free of the compound having the structure:


19. The method of claim 18, wherein the solvent is selected from thegroup consisting of water, methanol, ethanol, isopropanol, diethylether, tetrahydrofuran, acetonitrile, acetone, ethyl acetate, n-heptane,hexanes and cyclohexane.
 20. The method of claim 18, wherein the solventcomprises water.
 21. The method of claim 20, wherein the method furthercomprises: washing the reaction mixture with acetonitrile tosubstantially remove the water.
 22. The method of claim 21, wherein themethod further comprises: forming a solution of the compound of FormulaVIII and methanol; adding the solution to ethyl acetate to precipitatethe compound of Formula VIII; washing the precipitated compound ofFormula VIII with n-heptane; and heating the precipitated compound ofFormula VIII at a temperature of from about 50° C. to about 100° C.,thereby forming the compound of Formula VIII having less than 5000 ppmof solvent.
 23. The method of claim 18, wherein the compound of FormulaVII is

or salts thereof is prepared by the method comprising: forming areaction mixture comprising a base, a solvent, a compound of Formula VI:

 and a compound having the structure:

under an inert atmosphere and without exposure to visible light, toprepare the compound of Formula VII, wherein R¹ and R^(1a) are eachindependently C₁₋₆ alkylene-SO₃H; R², R^(2a), and R³ are eachindependently selected from the group consisting of H, C₁₋₆ alkyl, andC₁₋₆ alkylene-SO₃H; R^(3a) is C₁₋₆ alkylene-COOH; each R⁴ and R^(4a) isindependently selected from the group consisting of H, C₁₋₆ alkyl,—SO₃H, and C₁₋₆ alkylene-SO₃H; R⁵ is SO₃H; and each subscript n is aninteger from 0 to
 4. 24. A method of preparing a compound of FormulaVIII having the structure:

the method comprising: forming a first reaction mixture comprisingtriethylamine, methanol, a compound of Formula I having the structure:

 and a compound of Formula II having the structure:

under conditions sufficient to prepare a compound of Formula V havingthe structure:

or salts thereof, the compound of Formula V having a purity of at least95% and substantially free of a compound of Formula IV having thestructure:

forming a second reaction mixture comprising triethylamine, acetic acid,acetonitrile, the compound of Formula V, and a compound of Formula IIIhaving the structure:

under an Argon atmosphere and without exposure to visible light, toprepare the compound of Formula VI having the structure:

or salts thereof; forming a third reaction mixture comprising sodiumcarbonate, dimethylformamide, the compound of Formula VI, and thecompound having the structure:

under an inert atmosphere and without exposure to visible light, toprepare the compound of Formula VII having the structure:

 and forming a fourth reaction mixture comprising sodium acetate, thecompound of Formula VII, and water, under conditions suitable to formthe compound of Formula VIII substantially free of the compound havingthe structure: